3 research outputs found
Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter
Transcription of the prostate-specific antigen (PSA) gene is androgen
regulated. The PSA promoter contains at position -170 the sequence
AGAACAgcaAGTGCT, which is closely related to the ARE (androgen response
element) consensus sequence GGTACAnnnTGTTCT. This sequence is a high
affinity androgen receptor (AR) binding site and acts as a functional ARE
in transfected LNCaP cells. A 35-base pair segment starting at -400 (ARR:
androgen response region; GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTG) cooperates
with the ARE in androgen induction of the PSA promoter. A construct with
three ARR copies linked to a minimal PSA promoter showed a strong
(104-fold) androgen induced activity. The ARR was also able to confer
androgen responsiveness to a minimal thymidine kinase promoter. Both AR
binding and transcriptional activity resided in a 20-base pair ARR
subfragment: CAGGGATCAGGGAGTCTCAC (2S). Mutational analysis indicated that
the sequence GGATCAgggAGTCTC in the 2S fragment is a functionally active,
low affinity AR binding site. Like AR, the glucocorticoid receptor was
able to stimulate PSA promoter activity. Both the ARE and ARR are involved
in dexamethasone regulation of the PSA promoter. Both the AR and
glucocorticoid receptor were 20-100-fold more active on ARR-PSA and
ARR-thymidine kinase promoter constructs in LNCaP cells than in other cell
types (COS, HeLa, Hep3B, and T47D cells), indicating (prostate) cell
specificity
An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter
Prostate-specific antigen (PSA) is expressed at a
high level in the luminal epithelial cells of the prostate
and is absent or expressed at very low levels in
other tissues. PSA expression can be regulated by
androgens. Previously, two functional androgenresponse
elements were identified in the proximal
promoter of the PSA gene. To detect additional,
more distal control elements, DNaseI-hypersensitive
sites (DHSs) upstream of the PSA gene were
mapped in chromatin from the prostate-derived
cell line LNCaP grown in the presence and absence
of the synthetic androgen R1881. In a region 4.8 to
3.8 kb upstream of the transcription start site of the
PSA gene, a cluster of three DHSs was detected.
The middle DNAseI-hypersensitive site (DHSII, at
;24.2 kb) showed strong androgen responsiveness
in LNCaP cells and was absent in chromatin
from HeLa cells. Further analysis of the region encompassing
DHSII provided evidence for the presence
of a complex, androgen-responsive and cellspecific
enhancer. In transient transfected LNCaP
cells, PSA promoter constructs containing this upstream
enhancer region showed approximately
3000-fold higher activity in the presence than in the
absence of R1881. The core region of the enhancer
could be mapped within a 440-bp fragment. The
enhancer showed synergistic cooperation with the
proximal PSA promoter and was found to be composed
of at least three separate regulatory regions.
In the center, a functionally active, high-affinity androgen
receptor binding site (GGAACATATTGTATC)
could be identified. Mutation of this element
almost completely abolished PSA promoter activity.
Transfection experiments in prostate and nonprostate
cell lines showed largely LNCaP cell specificity
of the upstream enhancer region, although
some activity was found in the T47D mammary
tumor cell line
A 6-kb promoter fragment mimics in transgenic mice the prostate-specific and androgen-regulated expression of the endogenous prostate-specific antigen gene in humans
Prostate-specific antigen (PSA) is a kallikrein-like serine protease,
which is almost exclusively synthesized in the luminal epithelial cells of
the human prostate. PSA expression is androgen regulated. Previously, we
characterized in vitro the proximal promoter, and a strong enhancer
region, approximately 4 kb upstream of the PSA gene. Both regions are
needed for high, androgen-regulated activity of the PSA promoter in LNCaP
cells. The goal of the present study is the in vivo characterization of
the PSA promoter. Three transgenic mouse lines carrying the Escherichia
coli LacZ gene, driven by the 632-bp proximal PSA promoter, and three
lines with LacZ, driven by the 6-kb PSA promoter, were generated.
Expression of the LacZ reporter gene was analyzed in a large series of
tissues. Transgene expression could not be demonstrated in any of the
transgenic animals carrying the proximal PSA promoter. All three lines
carrying the 6-kb PSA promoter showed lateral prostate-specific
beta-galactosidase activity. Transgene expression was undetectable until 8
weeks after birth. Upon castration, beta-galactosidase activity rapidly
declined. It could be restored by subsequent androgen administration. A
search for mouse PSA-related kallikrein genes expressed in the prostate
led to the identification of mGK22, which was previously demonstrated to
be expressed in the submandibular salivary gland. Therefore, the 6-kb
PSA-LacZ transgene followed the expression pattern of the PSA gene in
humans, which is almost completely prostate-specific, rather than that of
mGK22 in mice. In conclusion, the 6-kb promoter fragment appears to
contain most, if not all, information for androgen regulation and prostate
specificity of the PSA gene